Gene therapy is a technique used to correct defective genes that are responsible for the development of many diseases. The most common approach to gene therapy involves the insertion of a normal gene into a location within the genome to replace a nonfunctional gene. A vector is often used to deliver the therapeutic, normal gene to the subject's target cells. One vector used in gene therapy is adeno-associated virus (AAV), which is particularly attractive because it is not currently known to cause disease (thereby causing a very mild immune response), infects dividing and non-dividing cells, and may incorporate its genome into that of the host cell. However, AAV gene therapy has been challenged by its inherent 5 kilobase (kb) viral packaging limit. To deliver a large therapeutic gene using AAV gene therapy, investigators have attempted different strategies such as truncating the gene itself and/or the use of shorter transcriptional regulatory elements (Flotte, 1993; Ostedgaard et al., 2005). These approaches have met with some success for certain disease genes, but they did not completely solve the problem of delivering a large gene, such as the full-length dystrophin coding sequence, using AAV.
Mutations in the dystrophin gene result in Duchenne muscular dystrophy (DMD), Becker muscular dystrophy (BMD) and X-linked dilated cardiomyopathy (XLDC). Currently, there is no cure for these devastating diseases, but progress in gene therapy has brought hope of curing these relentless diseases. Dystrophin is a 427 kD rod-shaped cytosolic protein located under the sarcolemma (Hoffman et al., 1987; Koenig et al., 1988). It has four functional domains including the N-terminal, rod, cysteine-rich (CR) and C-terminal domains, as shown in FIG. 1. The rod domain can be further divided into 24 spectrin-like repeats and 4 proline-rich hinges. The N-terminal domain and a stretch of basically charged repeats in the rod domain connect dystrophin to γ-actin (Ervasti, 2007). The end of hinge 4 and the CR domain link dystrophin to the transmembrane protein dystroglycan (DG), which then binds to laminin in the extracellular matrix (Huang et al., 2000; Jung et al., 1995). The dystrophin-DG-laminin linkage is further stabilized by sarcoglycans (SGs) and sarcospan (SS). Essentially this structural link (between the cytoskeleton and the extracellular matrix) provides mechanical support to maintain sarcolemma integrity during muscle contraction. In dystrophin deficient muscle, the failure to absorb contraction-induced stress contributes to muscle pathology (Davies et al., 2006; Ervasti, 2007; Petrof, 1998; Petrof, 2002). The dystrophin C-terminal domain binds to two molecules of syntrophin (Syn) and one molecule of dystrobrevin (Dbr). Syntrophin and dystrobrevin also bind to each other (Peters et al., 1997; Sadoulet-Puccio et al., 1997). Both syntrophin and dystrobrevin have multiple isoforms. The predominant forms in muscle are α-syntrophin and α-dystrobrevin, respectively. Syntrophin is an important scaffolding molecule (Wang et al., 2000). Its PDZ-domain interacts with signaling molecule nNOS, as shown in FIG. 1 (Brenman et al., 1996; Hillier et al., 1999; Tochio et al., 1999). Together, dystrophin and its associated proteins (including DG, SG, sarcospan, syntrophin, dystrobrevin, and nNOS) form the dystrophin-associated glycoprotein complex (DGC) (Ervasti, 2007; Ervasti et al., 1991). The DGC provides mechanical support and signaling function for muscle.
In DMD patients, a complete loss of the dystrophin protein leads to severe muscle disease and premature death. DMD is the most prevalent lethal childhood genetic disease, affecting one in 3,500 newborn boys worldwide. In BMD patients, reduced expression or expression of a truncated dystrophin causes a relatively mild phenotype with later onset and relatively slow progression (Beggs et al., 1991; Bulman, 1991; Emery, 1991; Hoffman, 1993; Hoffman et al., 1988; Koenig et al., 1989). Selective loss of dystrophin in the heart leads to XLDC.
Gene therapy for DMD, BMD and XLDC attempts to replace the defective dystrophin gene with a functional dystrophin gene, so that all affected muscles in a patient's body may be treated. Currently, three gene therapy approaches have entered clinical trials with limited success. These therapies include antisense oligonucleotide (AON) mediated exon skipping, full-length dystrophin replacement with a plasmid vector, and adeno-associated virus (AAV) mediated microgene therapy.
AON-mediated exon-skipping aims at restoring the open reading frame by re-directing dystrophin RNA splicing. As defined by its mechanism, this approach only produces an internally truncated protein instead of the full-length protein. AON-mediated exon-skipping faces several challenges. First, it requires an accurate molecular diagnosis. The AON tailored to one type of mutation may not be applicable to other mutations. Second, the exon-skipping method cannot treat patients who carry mutations in the CR domain. Third, exon-skipping often requires repeated delivery of AON to the target tissue. It is currently not clear whether repeated AON administration is associated with untoward clinical effects.
Plasmid-mediated gene therapy has also met its challenges. The first clinical trial with the plasmid vector was reported in 2004 using direct muscle injection (Romero et al., 2004), but studies have demonstrated relatively low and variable dystrophin expression from the plasmid vector (Wolff et al., 2005). Currently the efficiency is below the therapeutic threshold (Romero et al., 2004). Additional hurdles to be resolved for the plasmid-mediated gene therapy include transient expression and poor cardiac transduction.
The AAV vector has several features that are extremely suitable for DMD, BMD and XLDC, which affects nearly all body muscles. First, AAV is an effective muscle gene delivery vector, as a single intravenous injection can result in widespread, systemic transduction in skeletal and heart muscles (Ghosh et al., 2007b; Gregorevic et al., 2004; Wang et al., 2005). Additionally, both skeletal and cardiac muscles are highly transduced by AAV vectors. The main drawback of the AAV vector is its 5 kb packaging capacity. The full-length dystrophin coding sequence is more than 11 kb, which is beyond the 5 kb packaging capacity of a single AAV vector.
Different approaches using dual-vector strategies have been attempted to expand AAV vector packaging capacity (Duan et al., 2006). Among these approaches are trans-splicing (ts) and overlapping (ov) AAV vector systems (Duan et al., 2000; Yan et al., 2000; Sun et al., 2000; Nakai et al., 2000; Duan et al., 2001 b). In tsAAV vectors, split segments of a target gene are engineered with splicing signals. Upon co-infection, the AAV genomes undergo head-to-tail recombination through the viral inverted terminal repeats (ITRs). The engineered splicing signals then remove the ITR junction and restore transgene expression. In the ovAAV vectors, the two split segments share a common sequence. Upon co-infection, these segments undergo homologous recombination to recover the full-length gene. The tsAAV and ovAAV strategies raised hope that AAV vector gene therapy could be expanded to diseases that are linked to large therapeutic gene. However, these approaches have failed solve the size problem, because inherent limitations related to the molecular properties of the target gene remain. Thus, the small packaging capacity is still considered a major limitation in AAV gene therapy (Flotte, 2007.).
Besides the plasmid vector therapy described above, other approaches to express the full-length dystrophin protein have been attempted without clinical success. For example, the vectors based on herpes simplex virus type 1 (HSV-1) amplicon and gutted adenovirus have been shown to have the capacity to express the full-length protein. However, HSV-1 amplicon approach has limited application for in vivo gene therapy because it cannot efficiently infect mature muscle (Wang, 2006). The gutted adenoviral vector approach also encounters problems. The gutted adenoviral vectors are the vectors deleted of all adenoviral sequences except for the terminal repeats and packaging signal. Compared with AAV vectors, the gutted adenoviral vectors are poorly expressed in skeletal muscle (Fechner et al., 1999; Nalbantoglu et al., 1999; Uchida et al., 2005), and some studies have also shown the vector genome loss over time (Dudley et al., 2004). Furthermore, the systemic whole body transduction has not been established with the gutted adenoviral vector.